Phasor-based FLIM analysis of NAD(P)H and FAD autofluorescence for label-free bacterial classification

Bacterial contamination poses significant risks, particularly in medical settings, where timely identification is crucial for effective treatment. Traditional diagnostic methods often fail to provide rapid results, underlining the need for alternative approaches.

We investigate the potential of fluorescence lifetime imaging microscopy (FLIM) for fast label-free classification of bacterial species based on their growth states and intrinsic fluorescence characteristics.

Utilizing two-photon excitation microscopy, we examined the fluorescence lifetimes of the endogenous fluorophores NAD(P)H and FAD in Escherichia coli K12, Staphylococcus aureus, and Pseudomonas fluorescens. Cells were studied in different growth states-exponential, cooled, and dead-and measurements were taken at excitation wavelengths of 740 and 900 nm. The FLIM data were processed using a multi-component exponential decay model and visualized using phasor plots and kernel density estimation.

Results revealed distinct fluorescence lifetime profiles for each species, with clear differences observed in the exponential growth phase. Notably, E. coli K12 and S. aureus could be distinguished in mixed samples using a single excitation wavelength and emission channel.

These findings demonstrate that FLIM-based metabolic imaging can distinguish between bacterial species without labels, providing a promising basis for rapid, high-resolution diagnostic tools in clinical and research settings. We underscore the potential of FLIM as a powerful tool for bacterial classification, offering advantages in speed and spatial resolution over traditional methods.